Introduction to LC/GC Instrumentation

1. Introduction to LC/GC Instrumentation CM3007 Introduction to Separation Science Analytical Chemistry Lecture #1

2. Milestones in Chromatography • 1903 Tswett - plant pigments separated on chalk columns • 1931 Lederer & Kuhn - LC of carotenoids • 1938 TLC and ion exchange • 1950 reverse phase LC • 1954 Martin & Synge (Nobel Prize) • 1959 Gel permeation • 1965 instrumental LC (Waters)

3. Basis of Chromatography • Definition: Cs = Cm K • Mechanism - selective retardation caused by interactions with bonded phase of stationary phase

4. Chromatogram • Detector signal vs. retention time or volume 1 2 Presence of several Peaks separated in Time/volume evidences Separation of components Detector Signal time or volume Peak area  Concentration

5. Definitions • Mobile phase - phase that moves through chromatograph • In LC - eluent • In GC - carrier gas • Stationary phase - column; phase that is stationary in chromatograph • Bonded phase - reactive groups imparted to stationary phase in order to achieve selectivity

6. Types of Chromatography • Classification by mobile phase: • Gas - Gas chromatography (GC) • 1951 Martin and James (fatty acids) • Liquid - Liquid chromatography (LC) • 1964 Horvath (Yale) instrument • 1966 Horvath and Lipsky (nucleic acid components) • Supercritical fluid - Supercritical fluid chromatography (SFC) • 1958 Lovelock (Yale)

7. Purpose of Chromatography • Identify components in mixtures • Isolate component in mixture • Demonstrate level of purity • Quantitation • internal standard analytical preparative or semi-preparative

8. Instrumentation for LC • Strong/weak eluent • Pump (up to 6000 psi) • Injector • Column • Detector • {Fraction Collector} • Computer

9. Instrumentation for GC • Carrier gas • N2, He, H2 • Injector • Column • Detector • Computer oven

10. Modes of LC Separation • Adsorption or normal phase • Reverse phase • Hydrophobic interaction • Size exclusion • Ion Exchange • Affinity

11. Modes of LC Separation • Adsorption or Normal phase • Stationary phase more polar than mobile phase • column: hydroxyapatite (Ca10(PO4)6(OH)2 • mobile phase: hexane, CH2Cl2, THF, CH3OH • gradient elution

12. Modes of LC Separation (cont’d) • Reverse phase (RPC) • Stationary phase hydrophobic and mobile phase hydrophilic • column: silica, polystyrene covalently modified with alkyl chain 3-18 C’s • mobile phase: buffered water + organic solvent (propanol CH3CN, CH3OH) • gradient elution • Affinity (AC) • Based on specific and selective interactions between enzymes or antibodies and ligands

13. Modes of LC Separation (cont’d) • Hydrophobic interaction (HIC) • Stationary phase hydrophobic and mobile phase hydrophilic • stationary phase: low density of short alkyl chains or phenyl • mobile phase: decreasing concentration of high salt solution (3M NH4SO4) • gradient elution

14. Modes of LC Separation (cont’d) • Size Exclusion (Gel Permeation) (SEC) • Chemical interactions between sample and stationary phase undesirable; separation accomplished by sieving mechanism with porous stationary phase • stationary phase: varies - silica • mobile phase: low ionic strength (100 mM) buffer or dilute acid (0.1% TFA) with CH3CN or isopropanol (20-50%) • isocratic elution

15. Modes of LC Separation (cont’d) • Ion-Exchange (IEC) • Ion exchange interactions between cationic or anionic analyte and stationary phase bearing opposite charge • stationary phase: polystryrene, silica modified with functional groups such as quaternary amines • mobile phase: buffer containing increasing concentration of salt (NaCl, MgCl2, K3PO4, NH4SO4)

16. Ion Exchange • Strong - charge of strong ion exchanger is essentially independent of pH • sulfonic acids (cation) • quaternary amines (anion) • Weak - charge of weak ion exchanger is pH dependent • carboxymethyl (CM) (cation) • primary, secondary, and tertiary amines, e.g., diethylaminoethyl (DEAE) (anion)

17. Anion Exchange - Proteins • Anion exchange - protein is acidicbonded phase eitherweak - DEAEstrong - quaternary amine

18. Cation Exchange - Proteins • Cation exchange - protein is basicbonded phase eitherweak - CMstrong - sulfonic

19. Elution Approaches • Isocratic - constant mobile phase composition • Gradient - variable mobile phase composition • step - change accomplished sharply at a defined point in time • continuous - change accomplished gradually over time

20. Modes of GC Separation • Packed • earliest • Solid particles either porous or non-porous coated with thin (1 m) film of liquid • 1 - 8 mm ID; 1 - 10 m length

21. Modes of GC Separation • Capillary (open tubular) • modern • Inner wall modified with thin (1 m) film of liquid • 0.1 - 0.5 mm i.d.; 10 - 50 m length • types: • porous layer open tubular (PLOT) • wall-coated open tubular (WCOT) • liquid coating

22. GC Liquid Phase • Low volatility • High bp • Chemically unreactive • Examples: • 1-squalene • Tetrahydroxyethylenediamine • Carbowax (polyethylene glycol)

23. Elution Analogies: GC vs. LC • Isothermal (GC)  Isocratic (LC) • Programmed temperature (GC)  Gradient (LC) • Raising column temperature (GC) • Decreases retention time • Sharpens peaks

24. 5 Properties of a Good Detector • Sensitivity • Response/ C • Selectivity • Universal or selective response • selectivity - ability to distinguish between species • Rapid response • Linearity - concentration range over which signal proportional to concentration • Stability with respect to noise (baseline noise) and time (drift)

25. Detectors for LC • UV-vis • PMT (single ) • PDA (simultaneous multi-  monitoring) • Fluorescence • Electrochemical • Amperometry • NMR • microcoil NMR: J. Sweedler (U. Illinois)

26. Detectors for GC • Electron capture (ECD) • radioactive • good for X-, NO2- and conjugated • Thermal conductivity (TCD) • change in resistance of heated wire • Flame ionization (FID) • destruction of combustible sample in flame produces measurable current • Fourier transform infrared (FTIR) • Mass spectrometry (MS)

27. MS Components • Ionization source • Analyzer • Differentiating characteristic m/z • Ion detector

28. Ionization Methods - Examples • Electron capture (EC) • 70 eV e-+ neutral molecule  energetic molecular ion • hard; fragmentation • Chemical ionization (CI) • Reagent ion + molecule  molecular ion + reagent ion • Reagent ion = He, OH- (water), CH5+ or CH3+ (CH4) • soft; less fragmentation

29. Ionization Methods - Examples (cont’d) • Electrospray (ESI) • generation of ions by desolvation or desorption of charged liquid droplets • Matrix Assisted Laser Desorption (MALDI) • ionization facilitated by laser irradiation of sample dissolved in an organic matrix • EX: sinapinic acid

30. Types of MS Analyzers • Quadrupole - most common • Ion trap • Time of Flight (TOF)

31. Two Operational Modes • Scan • Collect mass data over known range • Slow • Selective ion monitoring (SIM) • Sample mass at predetermined values • Fast

32. Chromatogram time of injection tr Detector Response Retention Time

33. Chromatogram - GC-MS vs. LC (UV) • x-axis • GC-MS - m/z • LC - retention time or volume • y-axis - detector response • GC-MS - % abundance • LC - Abs